Induction and inhibition of cytochrome P450 and drug-metabolizing enzymes by climbazole

To determine the effect of climbazole on hepatic microsomal cytochrome P450 (P450) and drug-metabolizing enzymes, four different P450 isoforms (CYP2B1, 3A2, 2E1, and 2C12) were examined in female Long-Evans rats. Treatment of rats with climbazole resulted in the induction of P450 content. Climbazole both induced and inhibited aminopyrine N-demethylase activity, but not erythromycin N-demethylase activity. Uridine 5'-phosphate (UDP)-glucuronosyl transferase and glutathione S-transferase activities were also increased with climbazole treatment. Immunoblot analyses revealed that climbazole induces CYP2B1 and CYP3A2 at the lower dose examined, but it failed to increase CYP2B1 at the higher dose. Northern blot analysis revealed that climbazole markedly increases P450 2B1 mRNA. These results indicate that climbazole induces and inhibits P450-dependent drug-metabolizing enzymes in vivo and may have the dose-differential effect on CYP2B1 in rat liver.     

Climbazole is a new potent inducer of rat hepatic cytochrome P450

We examined the effect of climbazole on the induction of rat hepatic microsomal cytochrome P450 (P450), and compared the induction potency with other N-substituted azole drugs such as clorimazole. We found that climbazole is found to be a potent inducer of rat hepatic microsomal P450 as clorimazole. Induced level of P450 by climbazole was almost similar in extent to clorimazole when compared with other imidazole drugs in a dose- and time-dependent manner. Parallel to the increase in P450, climbazole increased aminopyrine and erythromycin N-demethylase, ethoxycoumarin O-deethylase, and androstenedione 16 beta- and 15 alpha/6 beta hydroxylase activities; however, clorimazole did not induce aminopyrine N-demethylase activity irrespective of its marked increase in P450 content. Immunoblot analyses revealed that climbazole induced CYP2B1, 3A2 and 4A1. The present findings indicate that climbazole is a new potent inducer of hepatic microsomal P450 and drug-metabolizing enzymes like clorimazole, but it may have some differential mechanism(s) for these enzymes' induction in rat liver.     

In vivo and in vitro induction of cytochrome P450 enzymes in beagle dogs.

The aim of this study was to determine the in vitro and in vivo effects of several prototypical inducers, namely beta-naphthoflavone, 3-methylcholanthrene, phenobarbital, isoniazid, rifampin, and clofibric acid, on the expression of cytochrome P450 (P450) enzymes in beagle dogs. For the in vitro induction study, primary cultures of dog hepatocytes were treated with enzyme inducers for 3 days, after which microsomes were prepared and analyzed for P450 activities. For the in vivo induction study, male and female beagle dogs were treated with enzyme inducers for 4 days (with the exception of phenobarbital, which was given for 14 days), after which the livers were removed and microsomal P450 activities were determined ex vivo. Treatment of male beagle dog hepatocyte cultures (n = 3) with beta-naphthoflavone or 3-methlychloranthrene resulted in up to a 75-fold increase in microsomal 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity, whereas in vivo treatment of male and female beagle dogs with beta-naphthoflavone followed by ex vivo analysis resulted in up to a 24-fold increase. Phenobarbital caused a 13-fold increase in 7-benzyloxyresorufin O-dealkylase (CYP2B11) activity in vitro and up to a 9.9-fold increase in vivo. Isoniazid had little or no effect on 4-nitrophenol hydroxylase activity in vitro. Rifampin caused a 13-fold induction of testosterone 6beta-hydroxylase (CYP3A12) activity in vitro and up to a 4.5-fold increase in vivo. Treatment of dogs in vivo or dog hepatocytes in vitro with clofibric acid appeared to have no effect on CYP4A activity as determined by the 12-hydroxylation of lauric acid. In general, the absolute rates (picomoles per minute per milligram of microsomal protein) of P450 reactions catalyzed by microsomes from cultured hepatocytes (i.e., in vitro rates) were considerably lower than those catalyzed by microsomes from dog liver (i.e., ex vivo rates). These results suggest that beagle dogs have CYP1A, CYP2B, CYP2E, and CYP3A enzymes and that the induction profile resembles the profile observed in humans more than in rats.     

Differential effects of 3 dipyridyl isomers on hepatic microsomal cytochrome P450 and heme oxygenase in rats

We compared the effects of 3 dipyridyl isomers, 2,2′-dipyridyl, 2,4′-dipyridyl and 4,4′-dipyridyl, on hepatic microsomal heme oxygenase and drug-metabolizing enzyme activities in male rats. 2,2′-Dipyridyl increased cytochrome P450 (P450) content at lower doses, but decreased with increasing dose levels. Immunoblot analysis revealed that 2,2′-dipyridyl did not induce both P450 1A1/2 and P450 2B1/2, in contrast to 2,4′- and 4,4′-dipyridyls, both of which were inducers of either P450 1A1/2 and/or P450 2B1/2. Some drug-metabolizing enzyme activities gradually declined with the increasing dose level of 2,2′-dipyridyl. 2,2′-Dipyridyl was able to induce hepatic microsomal heme oxygenase in a dose-dependent manner, but 2,4′- and 4,4′-dipyridyls did not, even at the highest dose (0.80 mmol/kg) examined. Treatment of rats with 2,2′-dipyridyl resulted in the increase of glutathione (GSH) content in a dose-dependent manner, but not 4-substituted isomers. A time course study with 2,2′-dipyridyl revealed that it produced a significant decrease in hepatic GSH content at early time periods (2–6 h) after its administration with an inverse increase in heme oxygenase activity. The present investigation has revealed that in contrast to the induction of P450 by 4-substituted dipyridyl compounds, 2,2′-dipyridyl is a novel inducer of hepatic microsomal heme oxygenase, together with the change in hepatic GSH content. This study would provide information on the differential effects of simple dipyridyl isomers on hepatic enzymes involved in heme and drug metabolism.     

Metabolite of 2,2',4',5-tetrabromobiphenyl, 3-methylsulphonyl-2,2',4',5-tetrabromobiphenyl,a potent inducer of CYP2B1/2 in rat

1. 3-Methylsulphonyl- and 4-methylsulphonyl-2,2',4',5-tetrabromobiphenyls (3-MeSO(2)- and 4-MeSO(2)-TetraBrBs) were detected in the liver, lung, kidney, adipose tissue and faeces of the 2,2',4',5-tetrabromobiphenyl (TetraBrB)-dosed rat. 2. The administration of 0.05-2.0 micromol kg(-1) doses of 3-MeSO(2)-TetraBrB produced corrresponding increases in the hepatic concentration of the methyl sulphone metabolite, corresponding increases in the content of total cytochrome P450, and corresponding increases in the activities of 7-benzyloxy-, 7-ethoxy- and 7-pentoxyresorufin O-dealkylases. The inducing effects of the 3-MeSO(2)-TetraBrB (0.2 micromol kg(-1)), both on the content of total P450 and on the activities of the three alkoxyresorufin O-dealkylases, were higher than that of the parent TetraBrB (342 micromol kg(-1)). 3. The major phenobarbital (PB)-inducible forms of P450, CYP2B1, CYP2B2, CYP3A2 and CYP2C6, were substantially induced by 3-MeSO(2)-TetraBrB, but CYP1A1 and CYP1A2 were not. On the other hand, the activities of drug-metabolizing enzymes and the four PB-inducible forms of P450 were unchanged by 4-MeSO(2)-TetraBrB treatment. 4. The induction profiles of these enzymes and P450 forms in rat treated with 3-MeSO(2)-TetraBrB were similar to those treated with PB. 5. The inducing ability of 3-MeSO(2)-TetraBrB (0.5 micromol kg(-1)) both on the activities of the three alkoxyresorufin O-dealkylases and on the contents of four PB-inducible forms of P450 was roughly equal to that of PB (431 micromol kg(-1) twice at a 24-h interval) or 3-MeSO(2)-2,2',4',5-tetrachlorobiphenyl (1 micromol kg(-1)). It is noteworthy that the effects of 3-MeSO(2)-TetraBrB on the drug-metabolizing enzymes CYP2B1 and CYP2B2 were several thousand-fold higher than those of parent TetraBrB, while the effect of its isomeric 4-MeSO(2)-TetraBrB were not. 6. The extent of hepatic accumulation of the 3-MeSO(2) metabolite after the administration of TetraBrB (342 micromol kg(-1)) was almost the same as that after the administration of 3-MeSO(2)-TetraBrB (0.1-0.2 micromol kg(-1)). The relationship between the hepatic concentration of the 3-MeSO(2) metabolite and the extent of enzyme induction after the administration of TetraBrB or 3-MeSO(2)-TetraBrB suggests that 3-MeSO(2)-TetraBrB plays an important role in the induction of microsomal drug-metabolizing enzymes by TetraBrB.     

Concurrent induction of rat hepatic microsomal cytochrome P450 and haem oxygenase by 2,2'-dipyridyl ketone: comparison with the effect of 2,2'-dipyridyl amine

1. The effect of 2,2'-dipyridyl ketone and 2,2'-dipyridyl amine on the induction of hepatic microsomal cytochrome P450 (P450) and heme oxygenase was compared, and their effects on five different P450 isoforms (P4501A1, 3A2, 2B1, 2E1 and 2C11) in rat were examined. 2. Treatment of rat with 2,2'-dipyridyL amine resulted in the marked induction of haem oxygenase to about seven-fold of the controls with a decrease in p450 content. 2,2'-Dipyridyl ketone produced concomitant induction of both P450 and haem oxygenase activity in a dose- and time-dependent manner without showing any sex differences. 3. Immunoblot analysis revealed that 2,2'-dipyridyl ketone slightly increased CYP2E1 and CYP3A2 at low doses, but not at high dose levels. There was no effect on P4502C11. P4502B1 was induced by the treatment with 2,2'-dipyridyl ketone in a dose-dependent manner. 4. These results indicate that dipyridyl compounds having different bridges between two aromatic moieties act as differential inducers of hepatic microsomal P450s and haem oxygenase.     

镉、铅、汞对苯巴比妥诱导肝微粒体药物代谢酶的影响

一次ip醋酸镉2.4mg/kg、醋酸铅100mg/kg或氯化汞 2.0 mg/kg均可抑制大鼠肝微粒体药物代谢酶。上述处理还可明显降低苯巴比妥对肝微粒’乙基吗啡N-脱甲基化酶、氨基比林N-脱甲基化酶、苯胺羟化酶和环己巴比妥羟化酶活力的诱导作用,降低苯巴比妥对细胞色素P450和细胞色素 b_5以及微粒体蛋白合成的诱导作用。结果提示镉、铅、汞可能通过降低微粒体酶的新生合成,抑制肝微粒体药物代谢酶。     

Induction of hepatic CYP2B1/2 by a teratogenic compound cis-1-[-4-(p-menthane-8-yloxy)phenyl]piperadine (YM9429) in rats

A teratogenic compound cis-1-[-4-(p-menthane-8-yloxy)phenyl]piperadine (YM9429) selectively induces skeletal malformations characterized by cleft palate in rat fetuses. In the present study, we investigated the effect of YM9429 on hepatic cytochrome P-450s and their activities in rats. Oral administrations of YM9429 at a dose of 250, 500 or 750 mg/kg daily for 3 days induced cytochrome P-450 contents in a dose-dependent manner. Concomitant induction of enzyme activities of benzphetamine N-demethylase, erythromycin N-demethylase and, to a lesser extent, aminopyrine N-demethylase was observed. Immunoblot analysis revealed that YM9429 up-regulated hepatic levels of CYP2B1/2 and CYP3A1/2 proteins. A single dose of YM9429 at 250 mg/kg induced CYP2B1/2 protein levels significantly. These results suggest that YM9429 is a strong inducer of cytochrome P-450 with characteristics resembling those of phenobarbital.     

Ginkgo biloba extract markedly induces pentoxyresorufin O-dealkylase activity in rats

We examined the effect of Ginkgo biloba extract (GBE) on hepatic drug-metabolizing enzymes, particularly cytochrome P450 (CYP), in rats. Rats were fed a GBE-containing diet or received GBE by intragastric gavage. The concentration of CYP and activity of various CYP enzymes in the liver were increased in a dose- and time-dependent manner. Significant increases in the concentration and activities of CYP enzymes were detected on day 1 of feeding of a 0.5% GBE diet and after administration of 10 mg GBE/kg body weight for 5 days by intragastric gavage. Among the CYP enzymes, the activity of pentoxyresorufin O-dealkylase (PROD), a CYP2B enzyme, was especially markedly increased. The induction of CYP2B enzyme by GBE was confirmed by Western blot analysis. Addition of GBE to a CYP assay system in vitro caused concentration-dependent inhibition of various CYP enzyme activities. The inhibition was more marked for the microsomal enzymes from GBE-treated rats than for those from control rats and more marked against PROD activity among the CYP enzymes tested. When the inhibition of various CYP enzymes activities by GBE in vitro was compared, no marked difference was observed between rat and human hepatic microsomal enzymes. These results indicate that excess intake of GBE induces CYP enzymes, particularly PROD, and may modify the efficacy of drugs taken simultaneously.     

Influence of propiverine on hepatic microsomal cytochrome p450 enzymes in male rats.

The bladder spasmolytics propiverine was shown to induce hepatic cytochrome P450 (P450) and aminopyrine and aniline oxidation in rats. To characterize the type of enzyme induction and its dose dependence, activities of seven hepatic microsomal P450-dependent monooxygenases were measured in 72 male LEW1A albino rats (body weight 236-295 g) after oral treatment with 0.5, 2, 6, and 60 mg/kg of propiverine hydrochloride for 5 days and compared with the effects of 40 mg/kg beta-naphthoflavone, 10 mg/kg phenobarbital, and 20 mg/kg dexamethasone (each group, n = 8). CYP2B expression was measured by Western blotting. Furthermore, the inhibitory potency of propiverine on P450 enzymes was evaluated in competition assays with three most specific monooxygenases. Results show that Propiverine induced several monooxygenases and CYP2B expression dose dependently. The effects were well comparable with a phenobarbital-type inducer with 60 mg/kg being equipotent to 10 mg/kg phenobarbital. Furthermore, propiverine in low concentrations inhibited pentylresorufin O-dealkylase (for CYP2B) in vitro. In conclusion, propiverine is a phenobarbital-type inducer on hepatic P450 enzymes in rats in doses about 100-times above the therapeutic doses in man.     

Comparison of effects of xenobiotics on extrahepatic and hepatic microsomal drug-metabolizing enzymes in mice

The content of microsomal protein is the same in both kidneys and small intestine, corresponding to 57% of the control value expressed as 100% in the untreated liver. The contents of P450 and cytochrome b(5), and the activity of NADPH-cytochrome c reductase in the kidney were higher than those in the small intestine, which were 17%, 22% and 41% of controls, respectively, in the former and 5%, 11% and 22% of controls in the latter. As compared with similar measurements made in the liver, the activities of substrate-metabolizing enzymes in these extrahepatic organs were very low. The activities of renal aniline hydroxylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, 7-methoxycoumarin O-demethylase and benzo(a)pyrene hydroxylase were 6%, 5%, 3%, 0.6% and 0.2% of controls, respectively. The activities of these enzymes in the small intestine were lower than those in the kidney or below the limits of detection. These results suggested that isoforms or their contents of P450 responsible for these substrate biotransformations are different among liver, kidneys and small intestine. Meantime, this study showed similar significant inductions by phenobarbital and rifampin of small intestinal and hepatic microsomal drug-metabolizing enzymes. In contrast, neither phenobarbital nor rifampin was capable of increasing renal microsomal enzymes, with the exception of benzo(a)pyrene hydroxylase which was induced by rifampin. These findings indicated that both liver and small intestine, but not kidneys contain the same phenobarbital- and rifampin-inducible P450 isoforms, cytochrome b(5) and NADPH-cytochrome c reductase. In addition, CCl(4) could be bioactivated by CYP2E1 to free radicals in the kidney which caused destruction of microsomal enzymes. In mice pretreated with phenobarbital, CCl(4) also attenuated the increase in content of P450 in the small intestine, which appeared to be a result of induction by phenobarbital of CYP2E1.     

Concurrent induction of rat hepatic microsomal cytochrome P450 and haem oxygenase by 2,2'-dipyridyl ketone: comparison with the effect of 2,2'-dipyridyl amine

1. The effect of 2,2'-dipyridyl ketone and 2,2'-dipyridyl amine on the induction of hepatic microsomal cytochrome P450 (P450) and heme oxygenase was compared, and their effects on five different P450 isoforms (P4501A1, 3A2, 2B1, 2E1 and 2C11) in rat were examined. 2. Treatment of rat with 2,2-dipyridyl amine resulted in the marked induction of haem oxygenase to about seven-fold of the controls with a decrease in P450 content. 2,2-'Dipyridyl ketone produced concomitant induction of both P450 and haem oxygenase activity in a dose- and time-dependent manner without showing any sex differences. 3. Immunoblot analysis revealed that 2,2'-dipyridyl ketone slightly increased CYP2E1 and CYP3A2 at low doses, but not at high dose levels. There was no effect on P4502C11. P4502B1 was induced by the treatment with 2,2'-dipyridyl ketone in a dose-dependent manner. 4. These results indicate that dipyridyl compounds having different bridges between two aromatic moieties act as differential inducers of hepatic microsomal P450s and haem oxygenase.     

Metabolic fate of pitavastatin (NK-104), a new inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase. Effects on drug-metabolizing systems in rats and humans

Pitavastatin (CAS 147526-32-7, NK-104) is a new and very potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and has been approved for treatment of hyperlipoproteinaemia. Pitavastatin has been studied for its effects on hepatic microsomal drug metabolism in rats, and the activities of several drug-metabolizing enzymes have been measured. No induction of the drug metabolizing enzymes (aniline hydroxylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase and UDP-glucuronic acid transferase) was found in the pitavastatin group compared to the control after the multiple administrations of pitavastatin at the dosage of 1-10 mg/kg per day for 7 days. Based on several different in vitro approaches, it is concluded that CYP2C9 is the enzyme responsible for the metabolism of pitavastatin and no metabolite is present in renal and intestinal microsomes. The CYP2C9 polymorphism was not involved in the pitavastatin metabolism. No inhibitory effect in CYP-mediated metabolism was detected on the tolbutamide 4-hydroxylation (CYP2C9) and testosterone 6 beta-hydroxylation (CYP3A4) in the presence of pitavastatin. The results suggested that pitavastatin did not affect the drug-metabolizing systems.     

Potential impact of steatosis on cytochrome P450 enzymes of human hepatocytes isolated from fatty liver grafts.

Liver grafts discarded for transplantation because of macrosteatosis can constitute a valuable source of human hepatocytes for in vitro metabolic and pharmacotoxicological studies or for therapeutic applications. A condition for using hepatocyte suspensions for these purposes is the preservation of their metabolic competence and, particularly, drug-metabolizing enzymes. A reduction in microsomal cytochrome P450 (P450) activities was observed in fatty livers (>40% steatosis) with respect to normal tissue. Similarly, decreased levels of 7-ethoxycoumarin O-deethylation and testosterone metabolism were observed in human hepatocyte cultures prepared from steatotic liver tissue. To clarify the potential impact of lipid accumulation on human hepatic P450 enzymes, we have used an in vitro model of "cellular steatosis" by incubation of cultured hepatocytes with increasing concentrations (0.25-3 mM) of long-chain free fatty acids (FFA). A dose-dependent accumulation of lipids in the cytosol is induced by FFA mixture. Hepatocytes exposed to 1 mM FFA for 14 h showed lower activity values of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 enzymes than nontreated hepatocytes (about 45-65% reduction). This treatment also produced significant decreases in CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 mRNA to about 55 to 75% of mRNA levels in control cells. Our results suggest that although human hepatocytes isolated from steatotic liver show reduced P450 activities, they are metabolically competent and can be used for drug metabolism studies.     

Stimulation of aniline, p-nitrophenol and N-nitrosodimethylamine metabolism in kidney by pyridine pretreatment of rabbits

Pyridine has been shown to cause liver and kidney damage in animals and in humans. In a previous study we examined the effects of pyridine on rabbit liver and lung microsomal drug-metabolizing enzymes. In this study, in vivo i.p. administration of pyridine to rabbits caused a significant 3.4-fold increase in kidney N-nitrosodimethylamine (NDMA) N-demethylase activity as compared to the activity in control rabbits. The same treatment also significantly stimulated the activity of other cytochrome P4502E1-associated enzymes. The activities of p-nitrophenol hydroxylase and aniline 4-hydroxylase in kidney microsomes were increased 4.9-and 4.5-fold, respectively. Pyridine treatment increased the P450 content of the kidney 1.6-fold (P<0.05). SDS-PAGE of both kidney and liver microsomes of pyridine-treated rabbits showed a protein band of enhanced intensity at 51,000 Mr migrating in the region of cytochrome P4502E1. p-Aminophenol, a 4-hydroxylation product of aniline, has been shown to be nephrotoxic and NDMA, a procarcinogen, has been shown to be carcinogenic following bioactivation by NDMA N-demethylase in a number of tissues including the kidney. Since pyridine was shown to be nephrotoxic, it is expected that pyridine potentiates the toxic and/or carcinogenic effects of aniline, p-nitrophenol and NDMA through induction of their metabolism by the cytochrome P450-dependent drug-metabolizing enzymes.     

The effect of aspartame on rat brain xenobiotic-metabolizing enzymes

This study demonstrates that chronic aspartame (ASP) consumption leads to an increase of phase I metabolizing enzymes (cytochrome P450 (CYP)) in rat brain. Wistar rats were treated by gavage with ASP at daily doses of 75 and 125 mg/kg body weight for 30 days. Cerebrum and cerebellum were used to obtain microsomal fractions to analyse activity and protein levels of seven cytochrome P450 enzymes. Increases in activity were consistently found with the 75 mg/kg dose both in cerebrum and cerebellum for all seven enzymes, although not at the same levels: CYP 2E1-associated 4-nitrophenol hydroxylase (4-NPH) activity was increased 1.5-fold in cerebrum and 25-fold in cerebellum; likewise, CYP2B1-associated penthoxyresorufin O-dealkylase (PROD) activity increased 2.9- and 1.7-fold respectively, CYP2B2-associated benzyloxyresorufin O-dealkylase (BROD) 4.5- and 1.1-fold, CYP3A-associated erythromycin N-demethylase (END) 1.4- and 3.3-fold, CYP1A1-associated ethoxyresorufin O-deethylase (EROD) 5.5- and 2.8-fold, and CYP1A2-associated methoxyresorufin O-demethylase (MROD) 3.7- and 1.3-fold. Furthermore, the pattern of induction of CYP immunoreactive proteins by ASP paralleled that of 4-NHP-, PROD-, BROD-, END-, EROD- and MROD-related activities only in the cerebellum. Conversely, no differences in CYP concentration and activity were detected in hepatic microsomes of treated animals with respect to the controls, suggesting a brain-specific response to ASP treatment.     

The effect of immunosuppressive agents (FK-506, rapamycin) on renal P450 systems in rat models.

It is well known that cyclosporin, rapamycin and FK-506 (tacrolimus) are metabolized by the liver microsomal cytochrome P450 enzyme system. Although there have been reports of interaction between these drugs and the renal P450 enzyme system, differences among these immunosuppressants has not been comprehensively demonstrated. We have studied the individual capacities of these immunosuppressants to induce renal microsomal P450 enzymes similar to CYP2B4 and CYP4A2 by examining renal function in treated rats, and have correlated the results by means of biochemical, immunological and immunohistochemical assays of renal P450 enzymes. Cyclosporin caused impairment of renal function with an increase in renal-specific P450 content, but FK-506 and rapamycin did not. Laurate omega- and (omega-1)-hydroxylase activity increased in rats treated with rapamycin but decreased in those treated with FK-506. Prostaglandin A1 (PGA1) omega-hydroxylase activity increased in rats treated with FK-506 but was reduced by treatment with cyclosporin. Aminopyrine N-demethylase activity increased in rats treated with cyclosporin or FK-506, but not in those treated with rapamycin. Western-blot analysis revealed significant induction of P450, (similar to CYP2B4 of the rabbit P450 isozyme) in kidneys from rats treated with cyclosporin but not in those from rats receiving FK-506 or rapamycin. Histochemical studies clearly demonstrated a form of P450 such as CYP4A2 in the proximal tubules of rats treated with cyclosporin, but not in those of rats treated with FK-506 or rapamycin. These results show that although cyclosporin has a strong effect on renal P450 systems and induces such a system in kidney cortex (microsomal P450), FK-506 and rapamycin have no substantial effect on the induction of renal P450. These findings might clarify the nephrotoxicity induced by these immunosuppressive drugs.     

Inhibitory and inducing effects of denzimol on carbamazepine metabolism in the rat

In vivo and in vitro alterations in carbamazepine (CBZ) metabolism and the extent of enzyme induction of the hepatic cytochrome P-450 system after chronic oral denzimol to rats were evaluated. No effect on drug-metabolizing enzymes was detected for this new anticonvulsant drug at a dose of 15 mg/kg, which is just above the anticonvulsive dose. At higher doses (60 mg/kg) denzimol significantly raised the hepatic cytochrome P-450 content, enhanced CBZ clearance and tend to shorten its elimination t1/2 and that of its active metabolite. These results, combined with those of a previous study showing impairment of CBZ metabolism after single doses of denzimol, suggest that the drug may have either inductive or inhibitory effects on microsomal mixed-function oxidase activity in the rat, depending on the dose and schedule of treatment.     

Characterization of the cytochrome P450 enzymes involved in the in vitro metabolism of ethosuximide by human hepatic microsomal enzymes

1. The relative roles of human hepatic cytochrome P450 (CYP) subfamilies participating in ethosuximide metabolism have been studied in vitro using humanized heterologous CYP microsomal systems expressing either CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1 or CYP3A4. 2. Ethosuximide was incubated with each expression system at 37 °C, and its hydroxylated metabolite was quantified by HPLC assay. 3. K m and V max values for metabolite formation were estimated for CYP3A4 and CYP2E1. The K m values for the metabolite formed were 1.40 and 0.24 mM for CYP3A4 and CYP2E1, respectively. The V max values were 0.65 and 0.14 nmol?mg ? 1 protein?min ? 1 for CYP3A4 and CYP2E1, respectively. 4. These parameters could not be measured for other enzymes, since metabolite concentrations formed were below the HPLC detection limits. 5. Immuno-inhibition studies using specific antibodies against CYP3A4 or CYP2E1 revealed that ethosuximide metabolite levels decreased when the amount of added CYP3A4 or CYP2E1 antibody increased, with anti-CYP3A4 antibodies yielding a greater inhibitory effect. 6. Simulations of scaled-up in vivo ethosuximide CL hepatic mediated by CYP3A4 and CYP2E1 based on the in vitro CL' int values, which were calculated from the foregoing respective V max and K m values, project that well over 90% of in vivo CL hepatic is due to CYP3A4. These results support an important role for CYP3A in human ethosuximide metabolism, and a minor role for CYP2E1.     

Simultaneous absolute protein quantification of transporters, cytochromes P450, and UDP-glucuronosyltransferases as a novel approach for the characterization of individual human liver: comparison with mRNA levels and activities

The purpose of the present study was to determine the absolute protein expression levels of multiple drug-metabolizing enzymes and transporters in 17 human liver biopsies, and to compare them with the mRNA expression levels and functional activities to evaluate the suitability of the three measures as parameters of hepatic metabolism. Absolute protein expression levels of 13 cytochrome P450 (P450) enzymes, NADPH-P450 reductase (P450R) and 6 UDP-glucuronosyltransferase (UGT) enzymes in microsomal fraction, and 22 transporters in plasma membrane fraction were determined using liquid chromatography/tandem mass spectrometry. CYP2C9, CYP2E1, CYP3A4, CYP2A6, UGT1A6, UGT2B7, UGT2B15, and P450R were abundantly expressed (more than 50 pmol/mg protein) in human liver microsomes. The protein expression levels of CYP3A4, CYP2B6, and CYP2C8 were each highly correlated with the corresponding enzyme activity and mRNA expression levels, whereas for other P450s, the protein expression levels were better correlated with the enzyme activities than the mRNA expression levels were. Among transporters, the protein expression level of organic anion-transporting polypeptide 1B1 was relatively highly correlated with the mRNA expression level. However, other transporters showed almost no correlation. These findings indicate that protein expression levels determined by the present simultaneous quantification method are a useful parameter to assess differences of hepatic function between individuals.